1. Field of the Invention
The present invention relates to an SPR sensor plate and an immune reaction measuring instrument using the SPR sensor plate, and in particular, to an SPR sensor plate utilizing a surface plasmon resonance (hereafter referred to as xe2x80x9cSPRxe2x80x9d) phenomenon and an immune reaction measuring instrument using this SPR sensor plate.
2. Description of the Related Art
In the field of biochemical analysis, immunoassays have commonly been used to detect a trace amount of protein in subjects. The immunoassay determines the concentration of a predetermined antigen in a subject on the basis of a specific immune reaction between the antigen (protein to be detected) and an antibody (produced using the antigen). With the immunoassay, the antigen to be detected can be measured without the necessity of isolation even if the subject contains plural types of antigens. This is the point where the immunoassay is different from chemical or physical measuring methods.
There are various immunoassays:
{circle around (1)} radio immunoassay: RIA method
{circle around (2)} enzyme immunoassay: EIA method
{circle around (3)} fluoro immunoassay: FIA method
Lately, the RIA method has not often been used due to its need to use isotopes. Additionally, the EIA method is now commonly used due to its ability to easily measure immune reaction. Furthermore, the FIA method is considered to be a sensitive and accurate measuring method. One of various EIA methods which uses a solid phase to measure an antibody is particularly called an xe2x80x9cELISA (enzymelinked immunosorbent assay)xe2x80x9d, and the ELISA further has the following two methods.
a. indirect method: using an antigen for the solid phase.
b. antibody capturing method: using an anti-Igm antibody for the solid phase.
These ELISA methods are used to determine the amount of an antibody for a particular pathogenic organism and an antibody for an allergen and to screen a monochronal antibody. A measuring kit used for the ELISA method is commonly a microplate having 96 recesses formed therein and on which immune reaction measurements are conducted. Accordingly, a larger number of subjects can be simultaneously measured, and many automated immune reaction measuring instruments have recently been on the market.
Many reagent makers are providing various reagents for the measuring kit for the ELISA method. For example, tPA is an enzyme that indirectly serves to dissolve fibrin present in the blood and which is associated with blood congelation or thrombus. In addition, PAI-1 is an enzyme that restrains the tPA to cause blood congelation or thrombus.
What is called an SPR sensor, used for immune reaction measuring instruments, is well known. The SPR sensor utilizes the surface plasmon resonance phenomenon and carries out measurements on the basis of the following principle: A thin metal film (gold, silver, or the like) of about 50 nm thickness is deposited on a bottom surface of a prism of a large refractive index. Then, predetermined light is allowed to enter the prism side at a critical angle or more toward the thin metal film. Since the thin metal film is translucent at about 50 nm, the light entering the prism side is transmitted through the thin metal film and reaches a surface of the thin metal film opposite to the prism to generate an evanescence field on this surface.
Surface plasmon resonance can be excited on the surface of the thin metal film by adjusting the light incident angle so that the number of waves in the evanescent field equals the number of waves in the surface plasmon resonance. In this case, the number of waves in the surface plasmon resonance depends on the dielectric constant of the thin metal film and on the refractive index of a subject fixed on the surface of the thin metal film opposite to the prism. Thus, the refractive index and dielectric constant of the subject can be determined. In this manner, the optical system and the subject are located opposite to each other across the thin metal film, so that this sensor can be constructed easily.
As an application of the above principle, an SPR sensor for immune reaction measuring instruments employing optical fibers has been developed (manufactured by BIACORE Co., Ltd.; trade name: BIACORE Probe). In this SPR sensor employing optical fibers, a clad is removed from an outer peripheral surface of a tip portion of the optical fiber and an end surface of the tip of the optical fiber is cut flat or polished and then coated with silver. Additionally, the outer peripheral surface of the tip of the optical fiber is coated with a thin metal film (gold or silver or the like). Furthermore, the thin metal film on the outer peripheral surface of the tip of the optical fiber is covered with dielectric film, on which an antibody for use in immune reaction measurements if fixed. Further, the optical fiber has a predetermined light source disposed on the other end side thereof so as to introduce light thereinto.
The immune reaction measuring method for the SPR sensor configured as described above will be described. First, light is introduced into the optical fiber and light of a predetermined wavelength excites the surface plasmon resonance at the tip portion of the optical fiber. The light wavelength exciting the surface plasmon resonance varies with the refractive indices of the dielectric film and the antibody. The intensity of the light of the wavelength that has induced the surface plasmon resonance attenuates. Thus, immune reaction can be measured by comparing the wavelength of the light attenuating most significantly before the immune reaction with that of the light attenuating most significantly after the immune reaction. In addition, an SPR sensor employing a prism in place of optical fibers has been developed.
The above conventional examples have the following disadvantages: If the SPR sensor is formed of an optical fiber, a thin metal film must be formed on an outer peripheral surface of a tip portion of a light waveguide of the optical fiber (for example, Au must be deposited thereon). Since, however, the optical fiber is fine, the thin metal film cannot be appropriately formed.
Additionally, for actual immune reaction measurements, an antibody must be bound to a surface of the thin metal film. Since, however, the tip portion of the light waveguide of the optical fiber is fine and cylindrical, it is disadvantageously difficult to fix the antibody thereto.
In addition, the prism type (using a single wavelength as a light source) carries out immune reaction measurements on the basis of the relationship between the light incident angle and the light intensity, so that it requires a drive section for varying the light incident angle. Consequently, it has a complicated structure.
An object of the present invention is to eliminate such disadvantages of the conventional examples to provide an SPR sensor plate which particularly allows the easy fixation of an antibody and the easy retention of a subject and which can be manufactured easily as well as an immune reaction measuring instrument using this SPR sensor plate.
To attain the above object, the present invention provides an SPR sensor plate comprising an optical waveguide for allowing light from a light source to pass therethrough and a sensing metal film formed on part of a surface of the optical waveguide, wherein the optical waveguide has reflecting metal films formed on opposite end surfaces thereof except for a light incident surface and a light exit surface, and at least one of the incident surface and exit surface is inclined through a predetermined inclination. With this configuration, light is incident on the light waveguide from the incident surface, and the incident light is repeatedly reflected inside the light waveguide. Then, while passing through the light waveguide, the light induces surface plasmon resonance on the sensing metal film. Subsequently, the light is emitted from the exit surface of the light waveguide and is incident on light detecting means. Accordingly, an optical path length producing the surface plasmon resonance is prolonged, and consequently, measuring sensitivity is improved. Additively, since the incident surface or the exit surface is inclined, it becomes easier to set the light source and an optical axis of the light detecting means.